The family Flaviviridae includes hemorrhagic fever viruses (e.g. dengue viruses, yellow fever virus) and encephalitis viruses (e.g. West Nile virus, Japanese encephalitis viruses). The long-term objective of this project is to determine the effect of flavivirus-specific CD4+ and CD8+ T cell responses induced by primary flavivirus infection on the immune response to subsequent infection with related flaviviruses. In Specific Aim 1, we will characterize effector responses, TCR avidity and signaling to homologous and heterologous virus CD4+ and CD8+ T cell epitopes in primary flavivirus-immune individuals. In Specific Aim 2, we will generate primary flavivirus CD4+ and CD8+ T cell responses to variant epitopes from flavivirus-naTve individuals and in human HLA transgenic mice. We will characterize cross-reactivity as in Aim 1 and will compare our findings in vitro to those generated in vivo. In Specific Aim 3, we will determine the biologic relevance of our in vitro findings. We will define variant specific responses in humans with sequential flavivirus infections. Using human HLA transgenic mice, we will model primary and secondary responses using peptide immunization followed by subclinical West Nile virus infection. Lastly, in collaboration with Project 2, we will determine the effect of variant T cell stimulation of human T cells on primary human endothelial cells. These studies will help to elucidate mechanisms of cross-protection and may give insights into disease pathogenesis.